This invention is a method for the purification of heparinase and other eliminases from F. heparinum.
Heparinase is an eliminase which cleaves heparin at alphaglycosidic linkages in heparin's major repeating unit: -&gt;4)-2-deoxy-2-sulfamino- -D-glucopyranose 6-sulfate-(1-&gt;4)-alpha-L-idopyranosyluronic acid 2-sulfate-(1-&gt;. Heparin is used clinically, both in vitro and in vivo, to inhibit blood coagulation. A mucopolysacchride with a wide range of molecular weights of up to 20,000, average molecular weight 13,500, heparin works by directly inhibiting thrombin and activated Factor X as well as other serine esterases in the blood.
The anticoagulant effect of heparin is neutralized clinically either by precipitation with protamine or as described in U.S. Ser. No. 044,245 entitled "Extracorporeal Reactors Containing Immobilized Species" filed May 22, 1987 by Howard Bernstein, et al., and U.S. Ser. No. 044,340 entitled "Bioreactor Containing Suspended, Immobilized Species" filed Jun. 6, 1987, by Lisa E. Freed, et al., reactors containing immobilized heparinase. The heparinase is immobilized to prevent leaching of the heparinase into the body via the blood passing through the reactor.
Sulfatase free heparinase, also designated catalytic grade heparinase, is required to completely remove the anticoagulant properties of heparin by enzymatic degradation. As described in U.S. Pat. No. 4,341,869 to Langer, et al., heparinase is produced by bacteria such as Flavobacterium heparinum. The organism is grown, the cells lysed, debris removed by centrifugation, and the cell extract passed through a hydroxylapatite, 3Ca.sub.3 (PO.sub.4).sub.2 or Ca(OH).sub.2 or Ca.sub.10 (PO.sub.4).sub.6 (OH).sub.2 column. A hydroxylapatite column can provide 10 to 100 fold enzyme enrichment when the protein is eluted from the column at high salt concentrations in a step-wise fashion. As described, higher yield of the enzyme is obtained by step-wise elution of the heparinase using a phosphate buffer solution of increasing sodium chloride concentration, ranging from 0.01M sodium phosphate pH 6.8 up to 0.10M sodium phosphate 0.19M sodium chloride pH 6.8.
This purification process was greatly improved by combining the hydroxylapatite chromatography with repeated gel filtration chromatography and chromatofocusing, as described by Yang, et al. in "Purification and Characterization of Heparinase from Flavobacterium heparinum" J.Biol.Chem. 260(3), 1849-1857 (1985).
The purified heparinase, a protein, has a molecular weight of 42,900.+-.1000 Daltons with a pI value of 8.5.
Although these methods are useful in preparing laboratory reagent quantities and characterizing the enzyme, they are inadequate for preparing heparinase in the quantity and the purity required for large scale clinical application. Additionally, the purification scheme outlined would be difficult to adapt to large scale recovery of the enzyme.
Other methods which have been used to extract proteins from the periplasmic space of Gram negative bacteria include osmotic shock treatment as the initial step. Typically these procedures include an initial disruption in osmotically stabilizing medium followed by selective release in non-stabilizing medium. The composition of these media (pH, protective agent) and the disruption methods used (chloroform, lysozyme, EDTA, sonication) vary among specific procedures reported. None of these has as yet been successfully applied to the purification of catalytic grade heparinase.
It is therefore an object of the present invention to provide a method for preparing highly pure heparinase in large quantities for use in commercial and clinical applications.
It is another object of the present invention to provide a method for isolation of other eliminases from F. heparinum.
It is a still further object of the invention to provide large quantities of purified, enzymatically active heparinase and other eliminases.